Protocol for preparing 16S ribosomal RNA gene amplicons for sequencing on the Illumina MiSeq system.
The protocol outlines a workflow that starts with amplicon PCR of the V3 and V4 regions of the 16S rRNA gene, followed by PCR clean‑up, dual‑index PCR, library quantification, normalization, pooling, denaturation and loading onto the MiSeq. It includes optional validation steps and guidance for using the MiSeq Reporter or BaseSpace for downstream analysis.\n\nThe document also provides detailed consumable lists, recommended laboratory practices to prevent PCR contamination, cleaning procedures using 0.5% sodium hypochlorite, and handling instructions for magnetic beads and temperature‑sensitive reagents.
| Expected size after Amplicon PCR | ~550 bp |
|---|---|
| Maximum libraries per pool | 96 |
| MiSeq run output | >20 million reads |
Up to 96 libraries can be pooled together using the Nextera XT index set.
The amplicon size after the first PCR is about 550 base pairs.
Libraries should be kept at temperatures ≤37 °C unless otherwise noted.